Mechanistic studies on the rat kidney flavoenzyme L-alpha-hydroxy acid oxidase.

The falvoenzyme L-alpha-hydroxy acid oxidase from rat kidney [T.H Cromartie and C.T. Walsh (1975), Biochemistry 14, 2588] fails to catalyze the elimination of HCl form D,L-beta-chlorolactate, although this compound is a substrate for oxidation by the enzyme. Deuterium isotope effects demonstrate that proton removal from the alpha carbon of alpha-hydroxy acids is fully rate limiting, a finding in agreement with observations on L-lactate dehydrogenase from yeast [F. Lederer (1974), Eur. J. Biochem. 46, 393] which also does not promote elimination from D,L-beta-chlorolactate. Both D-alpha-hydroxy acid oxidase were found to be rapidly and irreversibly inactivated by the acetylenic substrate 1-hydroxy-3-butynoate. The partially purified dehydrogenase was observed to be inactivated within 10 min by 6.8 times 10(-8) M hydroxybutynoate. For the more extensively studied oxidase, inactivation was found to occur after 25 catalytic events, inactivation occurring by covalent addition of the inactivator to the coenzyme. A stoichimometry of one molecule of hydroxybutynoate per flavine was found, and the time course of inactivation was unaffected by the presence of thiols. The oxidase could also be inactivated by prolonged incubation of the enzyme with 2-hydroxy-3-butenoate, and inactivation which could be completely prevented by the presence of thiolds. Since the inactivation with hydroxybutenoate also left the flavine coenzyme unaltered, the inactivation was attributed to Michael addition of nucleophiles on the enzyme of the ketobutenoate product. Several 4-alkyl-substitued 2-hydroxy-3-butynoates were also observed to inactivate the oxidase by both coenzyme modification and random addition to the apoenzyme. It is proposed that the inactivation may occur by nucleophilic addition of a C4 allenic carbanion to the oxidized flavine coenzyme.     

Ultrastructural localization of D-amino acid oxidase in microperoxisomes of the rat nervous system.

A recently developed procedure for the localization of D-amino acid oxidase (D-AAO) has been used to investigate the distribution of this enzyme in rat nervous tissue. Initial studies were carried out on kidney to validate the methods. The cytochemically demonstrable enzyme in kidney is inhibited by kojic acid, a known competitive D-AAO inhibitor. Omission of the catalse inhibitor, aminotriazole, from the cytochemical medium produces a marked diminution of D-AAO reaction product in kidney peroxisomes. This would be expected if catalase and D-AAO are present in the same particles. In brain, kojic acid-inhibitable D-AAO is demonstrable in numerous bodies within astrocytes especially in the cerebellum, a brain region known from biochemistry to contain particularly high levels of the oxidase. In preparations incubated for catalase, far fewer positive bodies are seen in the cerebellum. Moreover, omission of aminotriazole has little evident effect on the D-AAO reaction. Thus, the oxidase-containing cerebellar bodies may be relatively poor in catalse. In contrast, several nervous system cell types that contain relatively numerous catalase-positive bodies, contain none with detectable D-AAO. Such heterogeneity of peroxisome enzyme content is in accord with reports from biochemical studies of brain.     

The subcellular localization of phytanic acid oxidase in rat liver

Peroxisomal disorders (Zellweger's syndrome, neonatal adrenoleukodystrophy, infantile Refsum's syndrome, rhizomelic chondrodysplasia) show a series of enzymatic defects related to peroxisomal dysfunctions. Accumulation of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) has been found in several of these patients, caused by a defect in the alpha-oxidation mechanism of this acid. The fact that the alpha-oxidation of phytanic acid is defective in the peroxisomal disorders as well as in classical Refsum's disease makes it likely that this oxidation normally takes place in the peroxisomes. A series of experiments preformed to localize the phytanic acid oxidase in subcellular fractions of rat liver show, however, that the alpha-oxidation of phytanic acid is a mitochondrial process. Free phytanic acid is the substrate, and the only cofactors necessary are ATP and Mg2+.     

Immunoelectron microscopic localization of D-amino acid oxidase in rat kidney and liver

Summary The intracellular localization ofd-amino acid oxidase in rat kidney and liver has been investigated using the indirect immunogold postembedding technique. Different fixation and embedding conditions for optimal preservation of antigenicity and fine structure have been tested. Immunolabelling was possible only in tissues embedded in polar resins (glycol methacrylate and Lowicryl K4M). In kidney the enzyme was demonstrable only in the peroxisomes of the proximal tubule, where it was associated with the peroxisome core. The enzyme was present in all the peroxisomes of the proximal tubule and appeared to be codistributed with catalase. Control experiments and quantitative analysis confirmed the specificity of thed-amino acid oxidase immunolocalization. All the other cells in kidney failed to demonstrate any labelling. In liver, the immunolabelling was present in the matrix of the hepatocyte peroxisomes, whereas no traces of the enzyme were found in the nucleoid. The intensity of the immunolabelling in liver peroxisomes was lower than in kidney. No specific labelling was observed in cells other than hepatocytes.     

Electron microscopic localization of monoamine oxidase in the rat liver.

Two methods have been employed to localize monoamine oxidase activity in the cells of rat liver, using either 2-(2'-benzothiazolyl)-5-stryl-3-(4'-phtalhydrazidyl) tetrazolium chloride (BSPT) or ferricyanide as electron acceptor. With both methods monoamine oxidase activity was found both in the inner and the outer mitochondral membrane, although the outer membrane appeared the most probable location. In addition the BSPT method but not the ferricyanide method, revealed monoamine oxidase activity in the endoplasmatic reticulum. The results obtained by the two methods have been compared and are discussed in view of available biochemical data on monoamine oxidase.     

Ultrastructural localization of plasma retinol-binding protein in rat liver

Immunocytochemical studies were carried out to examine the subcellular localization of plasma retinol-binding protein (RBP) in rat liver. The studies used normal, retinol-deficient, and retinol-repleted retinol-deficient rats with or without colchicine pretreatment. Affinity-purified monomeric Fab' fragments from the IgG fraction of rabbit anti-rat RBP were conjugated to horseradish peroxidase. This conjugate effectively penetrated into tissue sections and enabled RBP to be localized by high resolution immunoelectron microscopy. In the normal liver parenchymal cell, RBP was found to be localized in the synthetic and secretory structures including endoplasmic reticulum (ER), Golgi complex (GC), and secretory vesicles. With the method used, significant localization of RBP was not observed in hepatic cells other than parenchymal cells. The distribution of RBP-positive areas within parenchymal cells changed markedly with retinol depletion. Thus, a heavy accumulation of RBP in the ER, accompanied by a marked decrease of the RBP-positive GC and secretory vesicles, was demonstrated in liver parenchymal cells from retinol-deficient rats. After repletion of deficient rats with retinol, the RBP that accumulated in the ER appeared to move rapidly from the ER through GC and secretory vesicles to the cell surface. Pretreatment with colchicine led to marked increase in RBP-positive secretory vesicles in retinol-repleted rat liver parenchymal cells. The results reported here demonstrate that the specific block in hepatic RBP secretion seen in retinol deficiency involves an inhibition of the movement of RBP from the ER to the GC in the parenchymal cell.     

Ultrastructural cytochemical localization of uricase in peroxisomes of rat liver

Ultrastructural localization of uricase (urate: oxygen oxidoreductase, E.C.1.7.3.3.) in rat liver parenchymal cells has been studied with the cerium technique. The cerous ions react with H2O2 generated by the activity of the enzyme in the presence of urate, forming the electron-dense reaction product of cerous perhydroxide. Tissue fixation is carried out by perfusion for 5 min with a low concentration (0.25%) of glutaraldehyde. Since in a biochemical assay it was found that the activity of uricase determined in Trismaleate buffer is substantially weaker than in the Pipes buffer, the classical medium of Briggs et al. (6) was modified, and the latter buffer was substituted for the Trismaleate. Vibratome sectons are incubated at 37 degrees C for 60 min in 0.1 M Pipes buffer, pH 7.8, containing 3 mM cerium chloride and 0.1 mM sodium urate. Under these conditions, the reaction product is localized in the crystalline cores of hepatic peroxisomes. The intensity of the staining is dependent on the concentration of the substrate and the incubation time. In control preparations incubated without urate or with 2,6,8-trichloropurine, a specific inhibitor of uricase, staining is almost completely abolished. In sections incubated with 5 mM cerium and 0.1 mM sodium urate, fine granules with a distribution corresponding to peroxisomes are also visible at the light microscopic level. This latter observation is invaluable for correlative light and electron microscopic studies.     

Immunocytochemical localization of d-amino acid oxidase in rat brain

d-amino acid oxidase (d-AAO) is a peroxisomal flavoenzyme, the physiological substrate and the precise function of which are still unclear. We have investigated D-AAO distribution in rat brain, by immunocytochemistry, with an affinity-purified polyclonal antibody. Immunoreactivity occurred in both neuronal and glial cells, albeit at different densities. Glial immunostaning was strongest in the caudal brainstem and cerebellar cortex, particularly in astrocytes, Golgi-Bergmann glia, and tanycytes. Hindbrain neurons were generally more immunoreactive than those in the forebrain. Immunopositive forebrain cell populations included mitral cells in the olfactory bulb, cortical and hippocampal neurons, ventral pallidum, and septal, reticular thalamic, and paraventricular hypothalamic nuclei. Within the positive regions, not all the neuronal populations were equally immunoreactive; for example, in the thalamus, only the reticular and anterodorsal nuclei showed intense labelling. In the hindbrain, immunopositivity was virtually ubiquitous, and was especially strong in the reticular formation, pontine, ventral and dorsal cochlear, vestibular, cranial motor nuclei, deep cerebellar nuclei, and the cerebellar cortex, especially in Golgi and Purkinje cells.     

Ultrastructural localization of xanthine oxidase activity in the digestive tract of the rat

Summary Precise localization of xanthine oxidase activity might elucidate physiological functions of the enzyme, which have not been established so far. Because xanthine oxidase is sensitive to chemical (aldehyde) fixation, we have localized its activity in unfixed cryostat sections of rat duodenum, oesophagus and tongue mounted on a semipermeable membrane. Previous studies had shown that this procedure enables the exact localization of activities of peroxisomal oxidases with maintenance of acceptable ultrastructure. Moreover, leakage and/or diffusion of enzyme molecules was prevented with this method. The incubation medium to detect xanthine oxidase activity contained hypoxanthine as substrate and cerium ions as capturing agent for hydrogen peroxide. After incubation, reaction product in the sections was either visualized for light microscopy or sections were fixed immediately and processed for electron microscopy. At the ultrastructural level, crystalline reaction product specifically formed by xanthine oxidase activity was found to be present in the cytoplasmic matrix of enterocytes and goblet cells and in mucus of duodenum. Moderate activity was found in the cytoplasm of apical cell layers of epithelia of oesophagus and tongue, with highest activity in the cornified layer. Moreover, large amounts of reaction product were found to surround bacteria present between cell remnants of the cornified layer of the oesophagus. Many bacteria surrounded by the enzyme showed signs of destruction and/or cell death. The intracellular localization of xanthine oxidase activity in the cytoplasm of epithelial cells as well as the extracellular localization suggest that the enzyme plays a role in the lumen of the digestive tract, for instance in the defence against microorganisms.     

Electron microscopic localization of monoamine oxidase in the rat liver

Summary Two methods have been employed to localize monoamine oxidase activity in the cells of rat liver, using either 2-(2′-benzothiazolyl)-5-stryl-3-(4′-phtalhydrazidyl) tetrazolium chloride (BSPT) or ferricyanide as electron acceptor. With both methods monoamine oxidase activity was found both in the inner and the outer mitochondral membrane, although the outer membrane appeared the most probable location. In addition the BSPT method but not the ferricyanide method, revealed monoamine oxidase activity in the endoplasmatic reticulum. The results obtained by the two methods have been compared and are discussed in view of available biochemical data on monoamine oxidase. Supported by research grants from the National Research Council of Canada (A 3651), The Swedish Medical Research Council (4145) and M. Bergwall's Foundation, Stockholm.     

Immunocytochemical localization of monoamine oxidase type B in rat liver

We used an immunohistochemical method to examine the localization of monoamine oxidase type B (MAOB) in rat liver. At the light microscopic level, MAOB was highly expressed in rat liver. It was intense around portal area, and weak around central area. All the hepatocytes examined had MAOB immunoreactivity. For the first time, using a double-labeling immunofluorescence histochemical method for laser microscopy, we report that no MAOB is found in endothelial cells, hepatic stellate cells, or Kupffer's cells. When examined under transmission electron microscopy, MAOB was localized to the mitochondrial outer membrane of hepatocytes. No apparent localization of MAOB was found in the rough endoplasmic reticulum, the crystal membrane of mitochondria, the nuclear envelope, or the plasma membrane.     

Ultrastructural localization of the mannose 6-phosphate receptor in rat liver

An affinity-purified rabbit antibody against rat liver mannose 6- phosphate receptor (MP-R) was prepared. The antibody was directed against a 215 kd-polypeptide and it recognized both ligand-occupied and free receptor. Anti-MP-R was used for immunofluorescence and immunoelectron microscopy of cryosections from rat liver. MP-R was demonstrated in all parenchymal liver cells, but not in endothelial lining cells. MP-R labeling was found at the entire plasma membrane, in coated pits and coated vesicles, in the compartment of uncoupling receptor and ligand, and in the Golgi complex. Lysosomes showed only scarce MP-R label. In double-labeling immunoelectron microscopy, MP-R co-localized with albumin in the Golgi cisternae and in secretory vesicles with lipoprotein particles. Cathepsin D was associated with MP- R in the Golgi cisternae. This finding indicates that MP-R/cathepsin D complexes traverse the Golgi complex on their way to the lysosomes. The possible involvement of CURL in lysosomal enzyme targeting is discussed.     

Ultrastructural localization of acid phosphatase in the neurohypophysis of the rat

Summary In the neurohypophysis of the rat, acid phosphatase is located in the lysosomes of pituicytes. These organelles contain lipid droplets which increase in number and size and distend them so that they lose their usual aspect while preserving their phosphatase activity.     

Immunocytochemical localization of acid phosphatase in rat liver

Localization of acid phosphatase (ACPase) in rat liver was investigated by immunocytochemical techniques. Rat liver was fixed by perfusion and cut into thick tissue slices, which were embedded in Epon or Lowicryl K4M. For light microscopy (LM), semithin Epon sections were stained for the enzyme ACPase by an indirect immunoenzyme technique. For electron microscopy (EM), ultra-thin Lowicryl K4M sections were stained by a protein A-gold technique. By means of LM, granular reaction deposits were observed in hepatocytes and sinus-lining cells. Stained granules were present in the juxtanuclear cytoplasm, but they did not correspond to a typical staining pattern for the Golgi complex. EM revealed that gold particles indicating ACPase antigens were present on lysosomes and on some vesicles locating in the trans Golgi region. Endosomelike vesicles were strongly positive for the labeling. Golgi cisterna were mostly negative, but weak signals were noted in dilated sacules. The plasma membranes on the sinusoidal and bile canalicular sides were labeled by a few gold particles. The results indicate that ACPase is present in endosomes and in a restricted area of plasma membrane, as well as in the lysosomal system.     

Ultrastructural localization of the ROMK potassium channel in rat liver and heart

The intracellular localization and distribution of the ROMK protein in rat liver and heart was studied by the electron microscopy of ultrathin sections using the antibodies against the ROMK channel protein, one of the contenders for the role of mitochondrial ATP-dependent potassium channel. In rat heart and liver tissues, the ROMK protein is localized on the membranes of mitochondrial cristae but differently distributed in hepatocytes and cardiomyocytes. In hepatocytes, colloidal gold particles were rather evenly distributed on the membranes of mitochondrial cristae. In cardiomyocytes, the number of granules was considerably lower than in hepatocytes, and they were also localized on the membranes of mitochondrial cristae and confined only by the center of these organelles.     

Subcellular localization of acid carboxypeptidase in rat liver and human fibroblasts.

The subcellular distribution of acid carboxypeptidase was investigated in rat liver, normal human skin (CRL 1501) and lung (WI-38) fibroblasts, galactosialidosis skin fibroblasts (GM 00806) and transformed lung fibroblasts (WI-38 VA 13). Results of differential and isopycnic centrifugations and osmotic activation experiments clearly indicate that the enzyme is located in lysosomes, in agreement with observations suggesting that carboxypeptidase is the protective protein of the 'Galjaard complex' which is defective in galactosialidosis.